Description: (Applicant's Description) The goal of this Laboratory Program is to design and synthesize nucleosides that can be cleaved by the new mutant enzymes created through the research described in this project and in Laboratory Programs 1 and 4. These prodrugs will be stable, and will not be substrates for other human or bacterial enzymes. These nucleoside prodrugs will then be used in combination with the appropriate enzyme in our gene therapy strategy . The specifics of our research efforts are as follows. We will collaborate with Laboratory Programs 1 and 4 in order to design new mutant enzymes derived from E. coli PNP . In tandem with the selection of mutant enzymes, we will work with Laboratory Program 4 to design, on the basis of current knowledge of E. coli PNP and the results of molecular modeling studies, new prodrugs that will be specifically paired with the mutant enzymes. Based upon our research to date, these prodrugs will be nucleosides with either 6-methylpurine or 2- fluoroadenine as the nitrogen base. We will synthesize the selected prodrugs, and they will be examined for substrate activity with the relevant mutant enzyme( s ), wild-type E. coli PNP , and any other relevant human enzymes (such as methylthioadenosine phosphorylase) in Laboratory Program 2. Promising compounds will be prepared in larger quantities for animal evaluations through Core B. In parallel, we will prepare a small number of new purines and examine their cytotoxicity profiles in Laboratory Program 2 to determine whether they are candidates for use in our prodrug strategy. This drug discovery process is by nature iterative, and at each step we will evaluate all the biological and structural data that we have and re-establish our priorities for future improvement ofboth the enzymes and the prodrugs that are paired with them. Our long-term goal will be to find an enzyme-prodrug combination that will be suitable for evaluation through an appropriate clinical trial.